Project Summary Complex organisms are able to rapidly induce select genes among thousands in response to diverse environmental cues. This occurs in the context of large genomes condensed with histone proteins into chromatin. The macrophage response to pathogen sensing, for example, rapidly engages highly conserved signaling pathways and transcription factors (TFs) for coordination of inflammatory gene induction. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a feature of inflammatory genes and, in general, dynamically regulated chromatin and transcription. The amino-terminal H3.3 `tail' differs from the other H3 proteins by a single amino acid, a serine at position 31. However, little is known of how (or which) features of H3.3, conserved from yeast to human, might enable rapid and high-level transcription. We have recently discovered a potent function for H3.3-specific histone phosphorylation (H3.3S31ph) in inflammatory gene transcription and surprising evidence that non-canonical activity of the DNA-damage response (DDR) pathway mediates this histone phosphorylation. Thus, we hypothesize that the DDR pathway is coopted for epigenetic regulation of inflammatory genes. In Aim 1 we will identify the factors and sequence of events that link DDR factors and H3.3S31ph to rapid inflammatory gene transcription and reveal the function of cross-talk between DDR and chromatin (H3.3S31ph) by employing novel histone mutant mouse models. Specifically, our experiments will enable us to distinguish between several candidate ?paths? to H3.3S31ph and amplification of transcription, including Topoisomerase dependency, and DNA break-dependent and -independent pathways. In Aim 2 we will identify how DDR-mediated H3.3S31ph uniquely regulates Pol II dynamics at select inflammatory genes to amplify their transcription. More generally, these studies will identify dedicated mechanisms that enable inflammatory gene induction with important implications for understanding inflammation and for informing more selective therapeutic strategies for diverse inflammatory diseases.